Allergic rhinitis and asthma are widespread conditions with complex and multifactorial etiologies. The severity of the conditions vary widely between individuals, and within individuals, dependent on factors such as genetics, environmental conditions, and cumulative respiratory pathology associated with duration and severity of disease. Both diseases are a result of immune system hyperresponsiveness to innocuous environmental antigens, with asthma typically including an atopic (i.e., allergic) component.
In asthma, the pathology manifests as inflammation, mucus overproduction, and reversible airway obstruction which may result in scarring and remodeling of the airways. Mild asthma is relatively well controlled with current therapeutic interventions including beta-agonists and low dose inhaled corticosteroids or cromolyn. However, moderate and severe asthma are less well controlled, and require daily treatment with more than one long-term control medication to achieve consistent control of asthma symptoms and normal lung function. With moderate asthma, doses of inhaled corticosteroids are increased relative to those given to mild asthmatics, and/or supplemented with long acting beta-agonists (LABA) (e.g., salmeterol) or leukotriene inhibitors (e.g., montelukast, zafirlukast). Although LABA can decrease dependence on corticosteroids, they are not as effective for total asthma control as corticosteroids (e.g., reduction of episodes, emergency room visits) (Lazarus et al., JAMA. 2001.285. 2583-2593; Lemanske et al., JAMA. 2001. 285: 2594-2603). With severe asthma, doses of inhaled corticosteroids are increased, and supplemented with both LABA and oral corticosteroids. Severe asthmatics often suffer from chronic symptoms, including night time symptoms; limitations on activities; and the need for emergency room visits. Additionally, chronic corticosteroid therapy at any level has a number of unwanted side effects, especially in children (e.g., damage to bones resulting in decreased growth).
Allergic rhinitis is inflammation of the nasal passages, and is typically associated with watery nasal discharge, sneezing, congestion and itching of the nose and eyes. It is frequently caused by exposure to irritants, particularly allergens. Allergic rhinitis affects about 20% of the American population and ranks as one of the most common illnesses in the US. Most suffer from seasonal symptoms due to exposure to allergens, such as pollen, that are produced during the natural plant growth season(s). A smaller proportion of sufferers have chronic allergies due to allergens that are produced throughout the year such as house dust mites or animal dander. A number of over the counter treatments are available for the treatment of allergic rhinitis including oral and nasal antihistamines, and decongestants. Antihistamines are utilized to block itching and sneezing and many of these drugs are associated with side effects such as sedation and performance impairment at high doses. Decongestants frequently cause insomnia, tremor, tachycardia, and hypertension. Nasal formulations, when taken improperly or terminated rapidly, can cause rebound congestion. Anticholinergics and montelukast have substantially fewer side effects, but they also have limited efficacy. Similarly, prescription medications are not free of side effects. Nasal corticosteroids can be used for prophylaxis or suppression of symptoms; however, compliance is variable due to side effects including poor taste and nasal irritation and bleeding. Allergen immunotherapy is expensive and time consuming and carries a low risk of anaphylaxis.
Persistent nasal inflammation can result in the development of nasal polyps. Nasal polyps are present in about 4.2% of patients with chronic rhinitis and asthma (4.4% of men and 3.8% of women) (Grigores et al., Allergy Asthma Proc. 2002, 23:169-174). The presence of polyps is increased with age in both sexes and in patients with cystic fibrosis and aspirin-hypersensitivity triad. Nasal polyposis results from chronic inflammation of the nasal and sinus mucous membranes. Chronic inflammation causes a reactive hyperplasia of the intranasal mucosal membrane, which results in the formation of polyps. The precise mechanism of polyp formation is incompletely understood. Nasal polyps are associated with nasal airway obstruction, postnasal drainage, dull headaches, snoring, anosmia, and rhinorrhea. Medical therapies include treatment for underlying chronic allergic rhinitis using antihistamines and topical nasal steroid sprays. For severe nasal polyposis causing severe nasal obstruction, treatment with short-term steroids may be beneficial. Topical use of cromolyn spray has also been found to be helpful to some patients in reducing the severity and size of the nasal polyps. Oral corticosteroids are the most effective medication for the short-term treatment of nasal polyps, and oral corticosteroids have the best effectiveness in shrinking inflammatory polyps. Intranasal steroid sprays may reduce or retard the growth of small nasal polyps, but they are relatively ineffective in massive nasal polyposis. Although nasal polyps can be treated pharmacologically, many of the therapeutics have undesirable side effects. Moreover, polyps tend to be recurrent, eventually requiring surgical intervention. Compositions and methods to inhibit post-surgical recurrence of nasal polyps are not presently available.
Other diseases characterized by similar inflammatory pathways include, but are not limited to, chronic bronchitis, pulmonary fibrosis, emphysema, chronic obstructive pulmonary disease (COPD), and pediatric asthma.
Interleukin Receptor 4-alpha and Inflammatory Signaling Pathways
It is generally acknowledged that allergy and asthma are a result of the dysregulation of the Th2 cytokine response. The presence of CD4+ T cells producing interleukin 4 (IL 4), IL 5 and IL 13 cytokines in bronchoalveolar lavage fluid and in airway epithelial biopsies of asthmatics has been clearly documented. Neutralization of IL 5 results in a decrease in eosinophilia in man, in the absence of a reduction in airway hyperresponsiveness (AHR). IL 4 and IL 13 have been implicated in multiple pathological processes that underlie asthma and allergy, including Th2 lymphocyte differentiation, induction of immunoglobulin E (IgE) production via regulation of the Ig isotype switch to the epsilon heavy chain in B lymphocytes, upregulation of IgE receptors and vascular associated adhesion molecule-1 (VCAM-1) expression, promotion of eosinophil transmigration in the lung, and mucus hypersecretion. IL 13 mediates the development of airway hyperresponsiveness (AHR) to cholinergic stimuli, lung remodeling, and promotion of the secretory phenotype of the inflamed airway epithelium. These observations make components of the Th2 cytokine pathway, particularly IL 4 and IL 13, potential targets for therapeutic intervention for asthma, allergy, and other forms of airway inflammation and/or hyperresponsiveness.
The IL 4 and IL 13 receptors share a common signaling chain, IL 4 receptor alpha (IL 4R-α). IL 4R-α pairs with the common gamma chain on cells of hematopoietic origin to form a type I IL 4R. This receptor binds exclusively IL 4. IL 4 and IL 13 also signal through a second receptor. The receptor is composed of IL 4R-α and IL 13R-α1 (type II IL 4R). IL 13R-α1 is present on both hematopoietic and non-hematopoietic cells. Formation of the IL 4R-α and IL 13R-α1 heterodimer results in a shift in affinity of IL 13R-α1 from a low affinity receptor, to a high affinity receptor. The IL 13R-α2 is a monomeric, high affinity IL 13 receptor that is thought to act as a decoy receptor to negatively regulate IL 13 activity. Signaling through the type I and type II IL 4Rs activates the Jak-Stat pathway; insulin-interleukin4 receptor (14R) motif associated factors such as insulin receptor substrate family of proteins; SH2 containing tyrosine phosphatases; and members of the Stat family such as Stat 6. A number of genetic studies have demonstrated that both IL 4R-α and Stat 6 are essential for allergen-induced pulmonary inflammation and AHR in mice.
IL 4R-α, was cloned independently by two groups (Galizzi et al., Int. Immunol., 1990, 2, 669-675; and Idzerda et al., J. Exp. Med., 1990, 171, 861-873). The human IL4 receptor gene was localized to 16p 11.2-16p 12.1 by in situ hybridization, and the mouse homolog was localized to the distal region of chromosome 7. The position on human chromosome 16 suggests that the IL4 receptor may be a candidate for rearrangements. For example, 12; 16 translocations are often associated with myxoid liposarcomas (Pritchard et al., Genomics, 1991, 10, 801-806).
Inhibitors of IL 4 and IL 13 independently have produced anti-inflammatory effects in mouse pulmonary inflammation models or in clinical trials (Wills-Karp M et al. Science 282: 2258-2261, 1998; Grunig G et al. Science 282: 2261-2263, 1998; Borish L C et al., Am J Respir Crit Care Med 160: 1816-1823, 1999; Kumar R K et al., Am J Respir Crit Care Med 170: 1043-1048, 2004; Yang G et al., Cytokine 28: 224-232, 2004) and are currently being pursued as novel therapeutics for allergy and asthma.
Antisense Oligonucleotides and Pulmonary Disease
Antisense oligonucleotides (ASOs) are being pursued as therapeutics for pulmonary inflammation, airway hyperresponsiveness, and/or asthma. Lung provides an ideal tissue for aerosolized ASOs for several reasons (Nyce and Metzger, Nature, 1997: 385:721-725, incorporated herein by reference); the lung can be targeted non-invasively and specifically, it has a large absorption surface; and is lined with surfactant that may facilitate distribution and uptake of ASOs. Delivery of ASOs to the lung by aerosol results in excellent distribution throughout the lung in both mice and primates. Immunohistochemical staining of inhaled ASOs in normalized and inflamed mouse lung tissue shows heavy staining in alveolar macrophages, eosinophils, and epithelium, moderate staining in blood vessels endothelium, and weak staining in bronchiolar epithelium. ASO-mediated target reduction is observed in dendritic cells, macrophages, eosinophils, and epithelial cells after aerosol administration. The estimated half life of a 2′-methoxyethoxy (2′-MOE) modified oligonucleotide delivered by aerosol administration to mouse or monkey is about 4 to 7, or at least 7 days, respectively. Moreover, ASOs have relatively predictable toxicities and pharmacokinetics based on backbone and nucleotide chemistry. Pulmonary administration of ASOs results in minimal systemic exposure, potentially increasing the safety of such compounds as compared to other classes of drugs.
Compositions and methods for formulation of ASOs and devices for delivery to the lung and nose are well known. ASOs are soluble in aqueous solution and may be delivered using standard nebulizer devices (Nyce, Exp. Opin. Invest. Drugs, 1997, 6:1149-1156). Formulations and methods for modulating the size of droplets using nebulizer devices to target specific portions of the respiratory tract and lungs are well known to those skilled in the art. Oligonucleotides can be delivered using other devices such as dry powder inhalers or metered dose inhalers which can provide improved patient convenience as compared to nebulizer devices, resulting in greater patient compliance.
Generally, the principle behind antisense technology is that an antisense compound hybridizes to a target nucleic acid and effects the modulation of gene expression activity, or function, such as transcription or translation. The modulation of gene expression can be achieved by, for example, target RNA degradation or occupancy-based inhibition. An example of modulation of target RNA function by degradation is RNase H-based degradation of the target RNA upon hybridization with a DNA-like antisense compound. Another example of modulation of gene expression by target degradation is RNA interference (RNAi) using small interfering RNAs (siRNAs). RNAi is a form of antisense-mediated gene silencing involving the introduction of double stranded (ds)RNA-like oligonucleotides leading to the sequence-specific reduction of targeted endogenous mRNA levels. This sequence-specificity makes antisense compounds extremely attractive as tools for target validation and gene functionalization, as well as therapeutics to selectively modulate the expression of genes involved in diseases.
Antisense oligonucleotides targeted to a number of targets including, but not limited to p38 alpha MAP kinase (US Patent Publication No. 20040171566, incorporated by reference); the CD28 receptor ligands B7-1 and B7-2 (US Patent Publication 20040235164, incorporated by reference); intracellular adhesion molecule (ICAM) (WO 2004/108945, incorporated by reference); and adenosine A1 receptor (Nyce and Metzger, Nature, 1997, 385.721-725) have been tested for their ability to inhibit pulmonary inflammation and airway hyperresponsiveness in mouse, rabbit, and/or monkey models of asthma when delivered by inhalation. Various endpoints were analyzed in each case and a portion of the results are presented herein. ASOs targeted to p38 alpha MAP kinase reduced eosinophil recruitment, airway hyperresponsiveness (AHR), and mucus production in two different mouse models. ASOs targeted to each B7.1 and B7.2 decreased target expression and eosinophil recruitment. An ASO targeted to B7.2 also reduced AHR. ASOs targeted to ICAM-1 decreased AHR and decreased neutrophil and eosinophil recruitment in mice. Treatment of Cynomolgus monkeys with an ASO targeted to ICAM-1 significantly reduced airway impedance (resistance) induced by methacholine challenge in naturally Ascaris allergen-sensitized monkeys. An ASO targeted to adenosine A1 receptor reduced receptor density on airway smooth muscle and reduced AHR in an allergic rabbit model. These data demonstrate that oligonucleotides are effectively delivered by inhalation to cells within the lungs of multiple species, including a non-human primate, and are effective at reducing airway hyperresponsiveness and/or pulmonary inflammation.
However, treatment with any ASO targeted to any inflammatory mediator involved in pulmonary inflammation is not always effective at reducing AHR and/or pulmonary inflammation. ASOs targeted to Jun N-terminal Kinase (JNK-1) found to decrease target expression in vitro were tested in a mouse model of asthma. Treatment with each of two different antisense oligonucleotides targeted to JNK-1 were not effective at reducing methacholine induced AHR, eosinophil recruitment, or mucus production at any of the ASO doses tested.
A number of ASOs and siRNAs designed to target IL 4R-α have been reported for use as research or diagnostic tools, or as pharmaceuticals for the treatment of respiratory disease. US Patent Application U.S.20030104410 teaches an array of nucleic acid probes useful as research tools to identify or detect gene sequences. Allelic variations in the IL 4R-α gene have been identified that increase receptor signaling (Hershey et al., NEJM, 1997, 337:1720-1725; Rosa-Rosa et al., J. Allergy Clin. Immunol. 1999, 104:1008-1014; Kruse et al., Immunol., 1999, 96, 365-371). PCT patent application WO 2000034789 teaches oligonucleotides for use in diagnostic testing to detect these allelic variations. Patent applications WO 2002085309, WO 2004011613 and U.S. 20040049022 teach ASOs targeted to a series of genes potentially relevant to respiratory disease, including IL 4R-α, for use in pharmaceutical compositions. Patent application U.S. 20050143333 teaches a series of siRNAs targeted to interleukins and interleukin receptors, including IL 4R-α. PCT application WO 2004045543 teaches algorithms and rational design and selection of functional siRNAs including those targeted to IL 4R-α. Although it is suggested in these publications that the ASOs and siRNAs can be used in pharmaceutical compositions, there are no data demonstrating the efficacy of the compounds in vivo for the prevention, amelioration, and/or treatment of any disease or disorder.